Oligonucleotides and methods for detection of west nile virus

ABSTRACT

The invention provides methods of detecting West Nile virus and oligonucleotide reagents derived from a West Nile virus consensus sequence that are useful in the methods of the invention.

This application is a divisional of application Ser. No. 10/985,805filed Nov. 10, 2004, which claims benefit of provisional applicationSer. No, 60/519,096 filed Nov. 12, 2003.

FIELD OF THE INVENTION

The present invention relates to methods and reagents for detecting WestNile virus. More particularly, the invention relates to nucleicacid-based methods of detecting West Nile virus and nucleic acidreagents useful in such methods.

BACKGROUND OF THE INVENTION

West Nile Virus is a spherical, enveloped virus containing asingle-stranded positive polarity RNA genome of approximately 11kilobases. West Nile Virus subtypes are distinguishable by antigenicvariations in the envelope (E or ENV) protein and by the presence of anN-glycosylation site (Asn-Tyr-Ser) at amino acids 154-156 (Jia, 1999,Lancet. 354; 1971-2). West Nile virus is taxonomically classified withinthe family Flaviviridae, genus Flavivirus. The virus was originallyisolated in 1937 from a febrile human who resided in the West NileDistrict of Uganda.

West Nile virus can be transmitted to humans and domestic animalsthrough mosquitoes and migratory birds that serve as amplifying hosts.Although West Nile virus infection is generally asymptomatic in areas ofthe world where the virus is endemic, infected humans can incur a mildfever, rash, nausea, headache, disorientation and back pain. Moreserious complications from West Nile virus infection include hepatitis,pancreatitis, encephalitis, myocarditis, meningitis, neurologicinfection, and death.

West Nile virus is geographically distributed in Africa, the MiddleEast, western and central Asia, India, Australia (Kunjin virus) andEurope. The first recorded epidemic occurred in Israel in the early1950's. More recently, outbreaks of human encephalitis caused by WestNile virus have been documented in Romania and Russia. West Nile virus,introduced recently into the northeastern United States, caused sevenhuman deaths in New York City and surrounding areas in 1999. Arelatively large number of birds, particularly crows, and horses alsodied. The subsequent recovery of West Nile virus from mosquitoes andbirds in 2000 confirmed that the virus had become established in thenortheastern United States. (Anderson et al. Proc. Nat'l Acad. Sci. USA98(23): 12885-12889, 2001).

In an attempt to prevent the spread of West Nile virus through controlof larval and adult mosquitoes, mapping, spraying and removal ofbreeding sites has been initiated in states where West Nile virus hasbeen identified. Despite these efforts, West Nile virus remains a threatdue to its mode of transmission. At present, there is no vaccine orother known treatment for West Nile virus infection. Accordingly thereremains an unmet need for reagents and methods for the diagnosis of WestNile virus infection.

During the 2002 epidemic of West Nile virus in the United States,twenty-three persons were reported to have acquired West Nile virusinfection after receipt of blood components from donors infected withthe virus (Morbidity and Mortality Weekly Report, 52(32): 769-772,2003). Consequently, there is also a need for reagents and tests fordiagnosing West Nile virus infection that can be used with blood orblood components such as plasma to identify infected blood.

Detection of West Nile virus using PCR-based assays has been reported.Russian patent application RU2199589, published Feb. 27, 2003, disclosesa PCR-based method for detection of West Nile virus in which a doubleamplification assay produces a 495 base pair PCR product that isanalyzed by agarose gel (from English language abstract).

WO 02/081511, published Oct. 17, 2002, assigned to Institute Pasteur andKimron Veterinary Institute, discloses a neuroinvasive and neurovirulentstrain of the West Nile virus, known as IS-98-ST1, nucleic acidmolecules derived from the genome thereof and methods of detecting WestNile virus.

Anderson et al., Science 286: 2331-2333, 1999 discloses primers foramplifying a 921 base portion of the genome of West Nile virus.Lanciotti et al., J. Clin. Microbiol. 38(11): 4066-4071 discloses a PCRassay for detecting West Nile virus wherein the primers were selectedfrom the envelope (ENV) and 3′ non-coding regions. Briese, T. et al.,Emerging Infectious Diseases 8(5) May 2002 discloses a PCR assay fordetecting West Nile virus wherein the primers were selected from the Egene. Huang et al. Emerging Infectious Diseases, 8(12) December 2002,discloses a PCR assay for detection of West Nile virus wherein theprimers were selected from the NS₅ (non-structural protein 5) and ENVgenes.

SUMMARY OF THE INVENTION

The invention provides methods and oligonucleotide reagents fordetecting West Nile virus.

The invention provides isolated oligonucleotides comprising

-   -   (a) R₁—N—R₂ wherein        -   N is an oligonucleotide selected from the group consisting            of

(SEQ ID NO: 2) 5′-CTGGATCGATGGAGAGGTGT-3′, (SEQ ID NO: 3)5′-TCCGGTCTTTCCTCCTCTTT-3′, (SEQ ID NO: 4) 5′-CTACCGTCAGCGATCTCTCC-3′,(SEQ ID NO: 5) 5′-TTCCTTTGCCAAATAGTCCG-3′, (SEQ ID NO: 6)5′-GACGTCGGGTCATTTGAAGT-3′, (SEQ ID NO: 7) 5′-ACTGCAATTCCAACACCACA-3′,(SEQ ID NO: 8) 5′-ATGTCCTGGATAACGCAAGG-3′, (SEQ ID NO: 9)5′-CTCCTCCAACTGCGAGAAAC-3′, (SEQ ID NO: 10) 5′-ATCGCGCTTGGAATAGCTTA-3′,(SEQ ID NO: 11) 5′-GACAGCCGTTCCAATGATCT-3′, (SEQ ID NO: 12)5′-AGGCCGGGTAGAGATTGACT-3′, (SEQ ID NO: 13) 5′-CCTGCAGCACCAATCTGTTA-3′,(SEQ ID NO: 14) 5′-CAGTGTTTATGGTGGCATCG-3′, (SEQ ID NO: 15)5′-GGCATCGTGATAAGCCATTT-3′, (SEQ ID NO: 16) 5′-TGGCAGAGCTTGACATTGAC-3′,(SEQ ID NO: 17) 5′-GCCGTTCTCTCAATCCACAT-3′, (SEQ ID NO: 18)5′-ATACTGGGGCAGTGTCAAGG-3′, (SEQ ID NO: 19) 5′-TAACGTTCTTGCCAGGTTCC-3′,(SEQ ID NO: 20) 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 21)5′-ACAGGTTGTAGTTCGGCACC-3′, (SEQ ID NO: 22) 5′-CCAGGCACTTCAGATCCATT-3′,(SEQ ID NO: 23) 5′-CTAGGCACAAACCAAACCGT-3′, (SEQ ID NO: 24)5′-GATTGACGCCAGGGTGTACT-3′, (SEQ ID NO: 25) 5′-ATGTCTTCCCCATGAAGTGC-3′,(SEQ ID NO: 26) 5′-CGCAGACAGACAACCAGCTA-3′, (SEQ ID NO: 27)5′-TTGACCTCAATTCTTTGCCC-3′, (SEQ ID NO: 28) 5′-GACGTCCCAGAATTAGAGCGC-3′,(SEQ ID NO: 29) 5′-TCCGGCTTCTCGTACTGTCT-3′, (SEQ ID NO: 30)5′-CTCTGTTTGGAACGCAACAA-3′, (SEQ ID NO: 31) 5′-GCCCCACCTCTTTTTAGTCC-3′,(SEQ ID NO: 32) 5′-AGTCGAGCTTCAGGCAATGT-3′, (SEQ ID NO: 33)5′-TGGTGTCTGAGTTGAGCAGG-3′, (SEQ ID NO: 34) 5′-TGAGTACAGTTCGACGTGGC-3′,(SEQ ID NO: 35) 5′-TTGAGAGGAGCCTGACCACT-3′, (SEQ ID NO: 36)5′-AGCTAAGGTGCTTGAGCTGC-3′, (SEQ ID NO: 37) 5′-ATGACGGTTCTTCCATCAGC-3′,(SEQ ID NO: 38) 5′-ACATCCAAGAGTGGAAACCG-3′, (SEQ ID NO: 39)5′-CGAGCTCTGCCTACCAATTC-3′, (SEQ ID NO: 40) 5′-GCAGGAGGAGAGTGGATGAC-3′,(SEQ ID NO: 41) 5′-TTCTCCACTGGGGTTTTGTC-3′, (SEQ ID NO: 42)5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 43) 5′-CCCTGACCTACAGCTTCAG-3′,(SEQ ID NO: 44) 5′-TAGTTCGCCTGTGTGAGCTG-3′, (SEQ ID NO: 45)5′-TTTTAGCATATTGACAGCCCG-3′, (SEQ ID NO: 46) 5′-TTGATTGGACTGAAGAGGGC-3′,(SEQ ID NO: 47) 5′-GCAATTGCTGTGAACCTGAA-3′, (SEQ ID NO: 48)5′-GCTGAAGCTGTAGGTCAGGG-3′, (SEQ ID NO: 49) 5′-CTGGTTGTGCAGAGCAGAAG-3′,(SEQ ID NO: 50) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 51)5′-GTCTCCTCTAACCTCTAGTCC-3′, (SEQ ID NO: 52) 5′-GCCACCGGAAGTTGAGTAGA-3′,(SEQ ID NO: 53) 5′-GAGACGGTTCTGAGGGCTTAC-3′, (SEQ ID NO: 54)5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′, (SEQ ID NO: 55)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, (SEQ ID NO: 56)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′, (SEQ ID NO: 58)5′-CAGGAGGACTGGGTTAACAAAGGCAAACCA-3′, and (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′;

-   -   -   -   R₁ is an oligonucleotide sequence of 0-20 contiguous                bases of the West Nile virus consensus sequence shown in                FIG. 1 (SEQ ID NO: 1) immediately upstream of the 5′ end                of N in said consensus sequence covalently linked to N                at the 5′ end, provided that when N is complementary to                said consensus sequence, R₁ is selected from the                complement of said consensus sequence; and            -   R₂ is an oligonucleotide sequence of 0-20 contiguous                bases of said consensus sequence immediately downstream                of the 3′-end of N in said consensus sequence covalently                linked to N at the 3′, end, provided that when N is                complementary to said consensus sequence, R₂ is selected                from the complement of said consensus sequence;

    -   (b) an isolated fragment of N as defined in (a) wherein said        fragment is 10-19 bases in length;

    -   (c) R₁—X—R₂, wherein X is at least 10 contiguous bases of N as        defined in (a), and R₁ and R₂ are as defined in (a), wherein        when R₁ is present, R₂ is absent and X is selected such that the        base at the 5′-end of X is the same as the base at the 5′-end of        N; and when R₂ is present, R₁ is absent and X is selected such        that the base at the 3′-end of X is the same as the base at the        3′-end of N;

    -   (d) an isolated oligonucleotide which has at least 80% sequence        identity with an oligonucleotide of (a), (b) or (c); or

    -   (e) an isolated oligonucleotide which is the full-length        complement of (a), (b), (c) or (d).

Preferably, the oligonucleotides of the invention comprise, or consistof, an oligonucleotide selected from the group consisting of

5′-CTGGATCGATGGAGAGGTGT-3′, (SEQ ID NO: 2) 5′-TCCGGTCTTTCCTCCTCTTT-3′,(SEQ ID NO: 3) 5′-CTACCGTCAGCGATCTCTCC-3′, (SEQ ID NO: 4)5′-TTCCTTTGCCAAATAGTCCG-3′, (SEQ ID NO: 5) 5′-GACGTCGGGTCATTTGAAGT-3′,(SEQ ID NO: 6) 5′-ACTGCAATTCCAACACCACA-3′, (SEQ ID NO: 7)5′-ATGTCCTGGATAACGCAAGG-3′, (SEQ ID NO: 8) 5′-CTCCTCCAACTGCGAGAAAC-3′,(SEQ ID NO: 9) 5′-ATCGCGCTTGGAATAGCTTA-3′, (SEQ ID NO: 10)5′-GACAGCCGTTCCAATGATCT-3′, (SEQ ID NO: 11) 5′-AGGCCGGGTAGAGATTGACT-3′,(SEQ ID NO: 12) 5′-CCTGCAGCACCAATCTGTTA-3′, (SEQ ID NO: 13)5′-CAGTGTTTATGGTGGCATCG-3′, (SEQ ID NO: 14) 5′-GGCATCGTGATAAGCCATTT-3′,(SEQ ID NO: 15) 5′-TGGCAGAGCTTGACATTGAC-3′, (SEQ ID NO: 16)5′-GCCGTTCTCTCAATCCACAT-3′, (SEQ ID NO: 17) 5′-ATACTGGGGCAGTGTCAAGG-3′,(SEQ ID NO: 18) 5′-TAACGTTCTTGCCAGGTTCC-3′, (SEQ ID NO: 19)5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20) 5′-ACAGGTTGTAGTTCGGCACC-3′,(SEQ ID NO: 21) 5′-CCAGGCACTTCAGATCCATT-3′, (SEQ ID NO: 22)5′-CTAGGCACAAACCAAACCGT-3′, (SEQ ID NO: 23) 5′-GATTGACGCCAGGGTGTACT-3′,(SEQ ID NO: 24) 5′-ATGTCTTCCCCATGAAGTGC-3′, (SEQ ID NO: 25)5′-CGCAGACAGACAACCAGCTA-3′, (SEQ ID NO: 26) 5′-TTGACCTCAATTCTTTGCCC-3′,(SEQ ID NO: 27) 5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-TCCGGCTTCTCGTACTGTCT-3′, (SEQ ID NO: 29) 5′-CTCTGTTTGGAACGCAACAA-3′,(SEQ ID NO: 30) 5′-GCCCCACCTCTTTTTAGTCC-3′, (SEQ ID NO: 31)5′-AGTCGAGCTTCAGGCAATGT-3′, (SEQ ID NO: 32) 5′-TGGTGTCTGAGTTGAGCAGG-3′,(SEQ ID NO: 33) 5′-TGAGTACAGTTCGACGTGGC-3′, (SEQ ID NO: 34)5′-TTGAGAGGAGCCTGACCACT-3′, (SEQ ID NO: 35) 5′-AGCTAAGGTGCTTGAGCTGC-3′,(SEQ ID NO: 36) 5′-ATGACGGTTCTTCCATCAGC-3′. (SEQ ID NO: 37)5′-ACATCCAAGAGTGGAAACCG-3′, (SEQ ID NO: 38) 5′-CGAGCTCTGCCTACCAATTC-3′,(SEQ ID NO: 39) 5′-GCAGGAGGAGAGTGGATGAC-3′, (SEQ ID NO: 40)5′-TTCTCCACTGGGGTTTTGTC-3′, (SEQ ID NO: 41) 5′-GGGTTAACAAAGGCAAACCA-3′,(SEQ ID NO: 42) 5′-CCGTGACCTACAGCTTCAG-3′, (SEQ ID NO: 43)5′-TAGTTCGCCTGTGTGAGCTG-3′, (SEQ ID NO: 44) 5′-TTTTAGCATATTGACAGCCCG-3′,(SEQ ID NO: 45) 5′-TTGATTGGACTGAAGAGGGC-3′, (SEQ ID NO: 46)5′-GCAATTGCTGTGAACCTGAA-3′, (SEQ ID NO: 47) 5′-GCTGAAGCTGTAGGTCAGGG-3′,(SEQ ID NO: 48) 5′-CTGGTTGTGCAGAGCAGAAG-3′, (SEQ ID NO: 49)5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50) 5′-GTCTCCTCTAACCTCTAGTCC-3′,(SEQ ID NO: 51) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

More preferably the oligonucleotides of the invention comprise anoligonucleotide selected from the group consisting of

5′-ATCGCGCTTGGAATAGCTTA-3′, (SEQ ID NO: 10) 5′-GACAGCCGTTCCAATGATCT-3′,(SEQ ID NO: 11) 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20)5′-ACAGGTTGTAGTTCGGCACC-3′, (SEQ ID NO: 21) 5′-GACGTCCCAGAATTAGAGCGC-3′,(SEQ ID NO: 28) 5′-TCCGGCTTCTCGTACTGTCT-3′, (SEQ ID NO: 29)5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 42) 5′-CCCTGACCTACAGCTTCAG-3′,(SEQ ID NO: 43) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50)5′-GTCTCCTCTAACCTCTAGTCC-3′, (SEQ ID NO: 51) 5′-GCCACCGGAAGTTGAGTAGA-3′,(SEQ ID NO: 52) and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

Most preferably the invention provides oligonucleotides comprising anoligonucleotide selected from the group consisting of

5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28) 5′-TCCGGGTTCTCGTACTGTCT-3′,(SEQ ID NO: 29) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

In other preferred embodiments, the invention provides oligonucleotidescomprising, or consisting of, an oligonucleotide selected from the groupconsisting of

(SEQ ID NO: 54) 5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′, (SEQ ID NO: 55)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, (SEQ ID NO: 56)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′, (SEQ ID NO: 58)5′-CAGGAGGACTGGGTTAACAAAGGCAAACCA-3′, and (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′.

More preferably, the oligonucleotide comprises

(SEQ ID NO: 56) 5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, or (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′.

The oligonucleotides of the invention can further comprise a detectablelabel. Preferably, the detectable label comprises a fluorescent moleculeattached at the 5′ end. More preferably, the oligonucleotides of theinvention further comprise a quencher molecule attached at the 3′ end.

Another aspect of the invention provides pairs of isolatedoligonucleotide sequences selected from the group consisting of

(a) 5′-CTGGATCGATGGAGAGGTGT-3′ (SEQ ID NO: 2) and5′-TCCGGTCTTTCCTCCTCTTT-3′ (SEQ ID NO: 3) (b) 5′-CTACCGTCAGCGATCTCTCC-3′(SEQ ID NO: 4) and 5′-TTCCTTTGCCAAATAGTCCG-3′ (SEQ ID NO: 5) (c)5′-GACGTCGGGTCATTTGAAGT-3′ (SEQ ID NO: 6) and 5′-ACTGCAATTCCAACACCACA-3′(SEQ ID NO: 7) (d) 5′-ATGTCCTGGATAACGCAAGG-3′ (SEQ ID NO: 8) and5′-CTCCTCCAACTGCGAGAAAC-3′ (SEQ ID NO: 9) (e) 5′-ATCGCGCTTGGAATAGCTTA-3′(SEQ ID NO: 10) and 5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO: 11) (f)5′-AGGCCGGGTAGAGATTGACT-3′ (SEQ ID NO: 12) and5′-CCTGCAGCACCAATCTGTTA-3′ (SEQ ID NO: 13) (g)5′-CAGTGTTTATGGTGGCATCG-3′ (SEQ ID NO: 14) and5′-GGCATCGTGATAAGCCATTT-3′ (SEQ ID NO: 15) (h)5′-TGGCAGAGCTTGACATTGAC-3′ (SEQ ID NO: 16) and5′-GCCGTTCTCTCAATCCACAT-3′ (SEQ ID NO: 17) (i)5′-ATACTGGGGCAGTGTCAAGG-3′ (SEQ ID NO: 18) and5′-TAACGTTCTTGCCAGGTTCC-3′ (SEQ ID NO: 19) (j)5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) and 5′ACAGGTTGTAGTTCGGCACC-3′(SEQ ID NO: 21) (k) 5′-CCAGGCACTTCAGATCCATT-3′ (SEQ ID NO: 22) and5′-CTAGGCACAAACCAAACCGT-3′ (SEQ ID NO: 23) (l)5′-GATTGACGCCAGGGTGTACT-3′ (SEQ ID NO: 24) and5′-ATGTCTTCCCCATGAAGTGC-3′ (SEQ ID NO: 25) (m)5′-CGCAGACAGACAACCAGCTA-3′ (SEQ ID NO: 26) and5′-TTGACCTCAATTCTTTGCCC-3′ (SEQ ID NO: 27) (n)5′-GACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) (o)5′-CTCTGTTTGGAACGCAACAA-3′ (SEQ ID NO: 30) and5′-GCCCCACCTCTTTTTAGTCC-3′ (SEQ ID NO: 31) (p)5′-AGTCGAGCTTCAGGCAATGT-3′ (SEQ ID NO: 32) and5′-TGGTGTCTGAGTTGAGCAGG-3′ (SEQ ID NO: 33) (q)5′-TGAGTACAGTTCGACGTGGC-3′ (SEQ ID NO: 34) and5′-TTGAGAGGAGCCTGACCACT-3′ (SEQ ID NO: 35) (r)5′-AGCTAAGGTGCTTGAGCTGC-3′ (SEQ ID NO: 36) and5′-ATGACGGTTCTTCCATCAGC-3′ (SEQ ID NO: 37) (s)5′-ACATCCAAGAGTGGAAACCG-3′ (SEQ ID NO: 38) and5′-CGAGCTCTGCCTACCAATTC-3′ (SEQ ID NO: 39) (t)5′-GCAGGAGGAGAGTGGATGAC-3′ (SEQ ID NO: 40) and5′-TTCTCCACTGGGGTTTTGTC-3′ (SEQ ID NO: 41) (u)5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) and 5′-CCCTGACCTACAGCTTCAG-3′(SEQ ID NO: 43) (v) 5′-TAGTTCGCCTGTGTGAGCTG-3′ (SEQ ID NO: 44) and5′-TTTTAGCATATTGACAGCCCG-3′ (SEQ ID NO: 45) (w)5′-TTGATTGGACTGAAGAGGGC-3′ (SEQ ID NO: 46) and5′-GCAATTGCTGTGAACCTGAA-3′ (SEQ ID NO; 47) (x)5′-GCTGAAGCTGTAGGTCAGGG-3′ (SEQ ID NO: 48) and5′-CTGGTTGTGCAGAGCAGAAG-3′ (SEQ ID NO: 49) (y)5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO: 51) (z)5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

More preferably the pair of isolated oligonucleotide sequences isselected from the group consisting of

(e) 5′-ATCGCGCTTGGAATAGCTTA-3′ (SEQ ID NO: 10) and5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO: 11) (j)5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) and 5′ACAGGTTGTAGTTCGGCACC-3′(SEQ ID NO: 21) (n) 5′-GACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) (u)5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) and 5′-CCCTGACCTACAGCTTCAG-3′(SEQ ID NO: 43) (y) 5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO: 51) (z)5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

Most preferably, the pair of isolated oligonucleotide is

5′-GACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) 5′-GCCACCGGAAGTTGAGTAGA-3′(SEQ ID NO: 52) and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

A further aspect of the invention provides sets of oligonucleotidesselected from the group consisting of

(aa) 5′-ATCGCGCTTGGAATAGCTTA-3′, (SEQ ID NO: 10)5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′ (SEQ ID NO: 54) optionally labeledwith a detectable label, and 5′-GACAGCCGTTCCAATGATCT-3′; (SEQ ID NO: 11)(bb) 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′ (SEQ ID NO: 55) optionally labeledwith a detectable label, and 5′-ACAGGTTGTAGTTCGGCACC-3′; (SEQ ID NO: 21)(cc) 5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′ (SEQ ID NO: 56) optionally labeledwith a detectable label, and 5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29)(dd) 5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 42)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′ (SEQ ID NO: 57) optionally labeledwith a detectable label, and 5′-CCCTGACCTACAGCTTCAG-3′ (SEQ ID NO: 43)(ee) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50)5′-CAGGAGGACTGGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 58) optionally labeledwith a detectable label, and 5′-GTCTCCTCTAACCTCTAGTCC-3′; (SEQ ID NO:51) (ff) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′ (SEQ ID NO: 59) optionally labeledwith a detectable label, and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO:53)

More preferably the set is

(cc) 5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′ (SEQ ID NO: 56) optionally labeledwith a detectable label, and 5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29)or (ff) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′ (SEQ ID NO: 59) optionally labeledwith a detectable label, and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO:53)

The invention additionally provides methods of detecting West Nile virusin a test sample comprising the steps of amplifying West Nile Virusnucleic acid in said test sample; and detecting amplified nucleic acid,wherein detection of amplified nucleic acid indicates the presence ofWest Nile virus in said test sample, wherein said amplifying step orsaid detecting step or both steps are performed with at least oneoligonucleotide of the invention. Preferably, the methods of theinvention are performed with a pair of oligonucleotides described above.More preferably, the methods of the invention are performed with a setof oligonucleotides described above. Most preferably, the methods of theinvention are performed with the oligonucleotide set

5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO:28)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′ (SEQ ID NO:56) optionally labeledwith a detectable label, and 5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO:29)or 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO:52)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′ (SEQ ID NO:59) optionally labeledwith a detectable label, and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO:53)

The invention provides other methods of detecting West Nile Virus in atest sample comprising the steps of hybridizing at least oneoligonucleotide of the invention with West Nile virus nucleic acid in atest sample; and detecting hybridization of said at least oneoligonucleotide of the invention with West Nile virus nucleic acid.

In preferred embodiments of the invention, the test sample compriseshuman blood plasma.

The invention also provides a method of identifying primers fordetection of a nucleic acid sequence comprising the steps of

-   -   (a) providing a nucleic acid sequence at least 1000 bases in        length;    -   (b) dividing said nucleic acid sequence into non-overlapping        segments approximately 500 bases in length starting from one end        of said sequence; and    -   (c) selecting forward and reverse primers each about 15-25 bases        in length from the sequence of at least one segment and/or its        complement, wherein the forward and reverse primers are selected        to have non-overlapping sequences and produce an amplicon having        from about 50 to 200 bases.

The invention also extends to primers identified by the aforementionedmethod.

Yet another aspect of the invention provides isolated oligonucleotidescomprising from about 15 to about 75 contiguous bases of the sequenceshown in FIG. 1 (SEQ ID NO: 1) or its complement, wherein theoligonucleotide binds with greater affinity to nucleic acid from NorthAmerican or Israeli West Nile virus isolates than West Nile virusisolates originating outside North America or Israel. Preferably, theoligonucleotides comprise from about 15 to about 50 contiguous bases ofthe West Nile virus consensus sequence shown in FIG. 1 (SEQ ID NO: 1) orits complement. More preferably, the oligonucleotides comprise fromabout 15 to about 25 contiguous bases of the West Nile virus consensussequence shown in FIG. 1 (SEQ ID NO: 1) or its complement. Still anotheraspect of the invention provides oligonucleotides as described abovethat bind with greater affinity to nucleic acid from North American orIsraeli West Nile virus isolates than West Nile virus isolatesoriginating outside the United States or Israel.

Another further aspect of the invention provides a test kit comprisingat least one oligonucleotide of the invention.

These and other aspects of the invention are more fully set out in theappended claims and the following Detailed Description.

BRIEF DESCRIPTION OF THE FIGURE

FIGS. 1A-1C show the West Nile virus consensus sequence (SEQ ID NO: 1).The consensus sequence was derived from sequence alignment andcomparisons of eight approximately full-length West Nile virus genomesfrom isolates identified in the United States (GenBank accessionnumbers, AF196835, AF260967, AF202541, AF206518, AF404753, AF404754,AF404755 and AF404756) using Vector Nti Suite 7.0 (InforMax, Frederick,Md., USA).

DETAILED DESCRIPTION OF THE INVENTION

The incidence of West Nile virus infection is increasing in the UnitedStates. West Nile virus rarely kills, but about one in 150 people whobecome infected with the virus will develop a potentially deadly case ofencephalitis or meningitis. There is no treatment or prevention of WestNile virus infection at present. Early detection is important fordiagnosing infection and treating symptoms. Detection of West Nile virusin donated blood is important to the prevention of infection resultingfrom contaminated blood or blood derivatives.

The present invention provides methods and oligonucleotide reagents fordetecting West Nile virus. The methods and oligonucleotides of theinvention are especially useful for detecting West Nile virus in bloodplasma.

The invention provides isolated oligonucleotides comprising R₁—N—R₂wherein N is an oligonucleotide selected from the group consisting of

(SEQ ID NO: 2) 5′-CTGGATCGATGGAGAGGTGT-3′, (SEQ ID NO: 3)5′-TCCGGTCTTTCCTCCTCTTT-3′, (SEQ ID NO: 4) 5′-CTACCGTCAGCGATCTCTCC-3′,(SEQ ID NO: 5) 5′-TTCCTTTGCCAAATAGTCCG-3′, (SEQ ID NO: 6)5′-GACGTCGGGTCATTTGAAGT-3′, (SEQ ID NO: 7) 5′-ACTGCAATTCCAACACCACA-3′,(SEQ ID NO: 8) 5′-ATGTCCTGGATAACGCAAGG-3′, (SEQ ID NO: 9)5′-CTCCTCCAACTGCGAGAAAC-3′, (SEQ ID NO: 10) 5′-ATCGCGCTTGGAATAGCTTA-3′,(SEQ ID NO: 11) 5′-GACAGCCGTTCCAATGATCT-3′, (SEQ ID NO: 12)5′-AGGCCGGGTAGAGATTGACT-3′, (SEQ ID NO: 13) 5′-CCTGCAGCACCAATCTGTTA-3′,(SEQ ID NO: 14) 5′-CAGTGTTTATGGTGGCATCG-3′, (SEQ ID NO: 15)5′-GGCATCGTGATAAGCCATTT-3′, (SEQ ID NO: 16) 5′-TGGCAGAGCTTGACATTGAC-3′,(SEQ ID NO: 17) 5′-GCCGTTCTCTCAATCCACAT-3′, (SEQ ID NO: 18)5′-ATACTGGGGCAGTGTCAAGG-3′, (SEQ ID NO: 19) 5′-TAACGTTCTTGCCAGGTTCC-3′,(SEQ ID NO: 20) 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 21)5′-ACAGGTTGTAGTTCGGCACC-3′, (SEQ ID NO: 22) 5′-CCAGGCACTTCAGATCCATT-3′,(SEQ ID NO: 23) 5′-CTAGGCACAAACCAAACCGT-3′, (SEQ ID NO: 24)5′-GATTGACGCCAGGGTGTACT-3′, (SEQ ID NO: 25) 5′-ATGTCTTCCCCATGAAGTGC-3′,(SEQ ID NO: 26) 5′-CGCAGACAGACAACCAGCTA-3′, (SEQ ID NO: 27)5′-TTGACCTCAATTCTTTGCCC-3′, (SEQ ID NO: 28) 5′-GACGTCCCAGAATTAGAGCGC-3′,(SEQ ID NO: 29) 5′-TCCGGCTTCTCGTACTGTCT-3′, (SEQ ID NO: 30)5′-CTCTGTTTGGAACGCAACAA-3′, (SEQ ID NO: 31) 5′-GCCCCACCTCTTTTTAGTCC-3′,(SEQ ID NO: 32) 5′-AGTCGAGCTTCAGGCAATGT-3′, (SEQ ID NO: 33)5′-TGGTGTCTGAGTTGAGCAGG-3′, (SEQ ID NO: 34) 5′-TGAGTACAGTTCGACGTGGC-3′,(SEQ ID NO: 35) 5′-TTGAGAGGAGCCTGACCACT-3′, (SEQ ID NO: 36)5′-AGCTAAGGTGCTTGAGCTGC-3′, (SEQ ID NO: 37) 5′-ATGACGGTTCTTCCATCAGC-3′,(SEQ ID NO: 38) 5′-ACATCCAAGAGTGGAAACCG-3′, (SEQ ID NO: 39)5′-CGAGCTCTGCCTACCAATTC-3′, (SEQ ID NO: 40) 5′-GCAGGAGGAGAGTGGATGAC-3′,(SEQ ID NO: 41) 5′-TTCTCCACTGGGGTTTTGTC-3′, (SEQ ID NO: 42)5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 43) 5′-CCCTGACCTACAGCTTCAG-3′,(SEQ ID NO: 44) 5′-TAGTTCGCCTGTGTGAGCTG-3′, (SEQ ID NO: 45)5′-TTTTAGCATATTGACAGCCCG-3′, (SEQ ID NO: 46) 5′-TTGATTGGACTGAAGAGGGC-3′,(SEQ ID NO: 47) 5′-GCAATTGCTGTGAACCTGAA-3′, (SEQ ID NO: 48)5′-GCTGAAGCTGTAGGTCAGGG-3′, (SEQ ID NO: 49) 5′-CTGGTTGTGCAGAGCAGAAG-3′,(SEQ ID NO: 50) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 51)5′-GTCTCCTCTAACCTCTAGTCC-3′, (SEQ ID NO: 52) 5′-GCCACCGGAAGTTGAGTAGA-3′,(SEQ ID NO: 53) 5′-GAGACGGTTCTGAGGGCTTAC-3′, (SEQ ID NO: 54)5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′, (SEQ ID NO: 55)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, (SEQ ID NO: 56)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′, (SEQ ID NO: 58)5′-CAGGAGGACTGGGTTAACAAAGGCAAACCA-3′, and (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′;

R₁ is an oligonucleotide sequence of 0-20 contiguous bases of the WestNile virus consensus sequence shown in FIG. 1 (SEQ ID NO: 1) immediatelyupstream of the 5′ end of N in the consensus sequence covalently linkedto N at the 5′ end, provided that when N is complementary to saidconsensus sequence, R₁ is selected from the complement of said consensussequence; and R₂ is an oligonucleotide sequence of 0-20 contiguous basesof the consensus sequence immediately downstream of the 3′-end of N inthe consensus sequence covalently linked to N at the 3′ end, providedthat when N is complementary to said consensus sequence, R₂ is selectedfrom the complement of said consensus sequence.

The oligonucleotides are selected by locating N in the consensussequence of FIG. 1 and continuing upstream in the 5′ direction from thefirst base of N and selecting 0-20 bases immediately adjacent to thefirst base of N and/or continuing downstream in the 3′ direction fromthe last base of N and selecting 0-20 contiguous bases immediatelyadjacent to the last base of N. If N is complementary to the consensussequence, R₁ and R₂ are selected in the same manner from the complementof the consensus sequence. Preferably, R₁ and R₂ are independently 0-15contiguous bases in length, more preferably 0-10 contiguous bases inlength, most preferably 0-5 contiguous bases in length.

The invention also provides isolated fragments of N as defined hereinwherein the fragment is 10-19 bases in length, preferably 15-19 bases inlength.

The invention further provides isolated nucleotides comprising R₁—X—R₂,wherein X is at least 10 contiguous bases of N as defined herein, and R₁and R₂ have the meanings defined herein, wherein when R₁ is present, R₂is absent and X is selected such that the base at the 5′-end of X is thesame as the base at the 5′-end of N; and when R₂ is present, R₁ isabsent and X is selected such that the base at the 3′-end of X is thesame as the base at the 3′-end of N. The oligonucleotides can beselected by locating N in the consensus sequence shown in FIG. 1 andselecting at least 10 contiguous bases beginning from the 5′-end andcontinuing for at least ten contiguous bases downstream towards the 3′end. Alternatively, the oligonucleotide can be selected by locating N inthe consensus sequence shown in FIG. 1 and selecting at least tencontiguous bases beginning from the 3′-end and continuing for at leastten contiguous bases upstream towards the 5′-end. When X is selectedfrom the 5′-end of N, R₂ is absent and R₁ is selected by continuingupstream in the 5′ direction from the first base of N and selecting 0-20bases immediately adjacent to the first base of N. When X is selectedfrom the 3′-end of N, R₁ is absent and R₂ is selected by continuingdownstream in the 3′ direction from the last base of N and selecting0-20 contiguous bases immediately adjacent to the last base of N.Preferably, R₁ and R₂ are independently 0-15 contiguous bases in length,more preferably 0-10 contiguous bases in length, most preferably 0-5contiguous bases in length. If N is complementary to the consensussequence, R₁ and R₂ are selected in the same manner from the complementof the consensus sequence.

Also provided are isolated oligonucleotides that have at least 80%sequence identity with an oligonucleotide having the formula R₁—N—R₂,wherein R₁, N and R₂ have the meanings defined herein, a fragment of Nas defined herein, or an oligonucleotide having the formula R₁—X—R₂,wherein R₁, X and R₂ have the meanings defined herein. Sequence identitycan be determined by manually aligning the sequences and calculating thepercentage of sequence identity from the number of bases that are thesame in both sequences or using a mathematical algorithm. Non-limitingexamples of such mathematical algorithms are the algorithm of Myers andMiller (1988) CABIOS 4:11-17; the local homology algorithm of Smith etal. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computer implementationsof these mathematical algorithms can be utilized for comparison ofsequences to determine sequence identity. Such implementations include,but are not limited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Version 8 (available from Genetics Computer Group(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using theseprograms can be performed using the default parameters. Preferably, theoligonucleotide has 85, 90 or 95% sequence identity with anoligonucleotide of the formula R₁—N—R₂, wherein R₁, N and R₂ have themeanings as defined herein, a fragment of N as defined herein, or anoligonucleotide having the formula R₁X—R₂ wherein R₁, X and R₂ have themeanings defined herein.

The invention further provides isolated oligonucleotides that are thefull-length complement of the oligonucleotides.

The term “isolated” oligonucleotide refers to an oligonucleotide that isfound in a condition other than its native environment. In a preferredform, the oligonucleotide is substantially free from other nucleic acidsequences, such as other chromosomal and extrachromosomal DNA and RNA,that normally accompany or interact with it as found in its naturallyoccurring environment. The term “isolated” oligonucleotide also embracesrecombinant oligonucleotides and chemically synthesizedoligonucleotides.

Another aspect of the invention provides methods of detecting West Nilevirus in a sample suspected of containing the virus comprising the stepsof amplifying West Nile Virus nucleic acid in a sample suspected ofcontaining such virus; and detecting amplified nucleic acid, whereindetection of amplified nucleic acid indicates the presence of West Nilevirus in the sample, which method uses at least one oligonucleotide ofthe invention to amplify or detect West Nile virus nucleic acid.

A further aspect of the invention provides methods of detecting WestNile virus in a sample comprising the steps of hybridizing at least oneoligonucleotide of the invention with West Nile virus nucleic acid anddetecting hybridization of the at least one oligonucleotide with theWest Nile virus nucleic acid.

West Nile virus is an RNA genome virus and it will be necessary in someassay formats to convert the RNA into DNA prior to amplification.Conversion of the RNA into DNA can be done using reverse transcriptase.

The amplifying step can be performed using any type of nucleic acidtemplate-based method, such as polymerase chain reaction (PCR)technology.

PCR technology relies on thermal strand separation followed by thermaldissociation. During this process, at least one primer per strand,cycling equipment, high reaction temperatures and specific thermostableenzymes are used (U.S. Pat. Nos. 4,683,195 and 4,883,202).Alternatively, it is possible to amplify the DNA at a constanttemperature (Nucleic Acids Sequence Based Amplification (NASBA) Kievits,T., et al., J. Virol Methods, 1991; 35, 273-286; and Malek, L. T., U.S.Pat. No. 5,130,238; T7 RNA polymerase-mediated amplification (TMA)(Giachetti C, et al. J Clin Microbiol 2002 July; 40(7):2408-19; orStrand Displacement Amplification (SDA), Walker, G. T. and Schram, J.L., European Patent Application Publication No. 0 500 224 A2; Walker, G.T., et al., Nuc. Acids Res., 1992; 20, 1691-1696).

Preferably, amplification is done using PCR and at least oneoligonucleotide primer selected from the consensus sequence or itscomplement. Primers are preferably 15-30 nucleotides long, morepreferably 15 to 25 bases long, most preferably, about twentynucleotides long. Preferably, the at least one primer is anoligonucleotide selected from the group consisting of

5′-CTGGATCGATGGAGAGGTGT-3′, (SEQ ID NO: 2) 5′-TCCGGTCTTTCCTCCTCTTT-3′,(SEQ ID NO: 3) 5′-CTACCGTCAGCGATCTCTCC-3′, (SEQ ID NO: 4)5′-TTCCTTTGCCAAATAGTCCG-3′, (SEQ ID NO: 5) 5′-GACGTCGGGTCATTTGAAGT-3′,(SEQ ID NO: 6) 5′-ACTGCAATTCCAACACCACA-3′, (SEQ ID NO: 7)5′-ATGTCCTGGATAACGCAAGG-3′, (SEQ ID NO: 8) 5′-CTCCTCCAACTGCGAGAAAC-3′,(SEQ ID NO: 9) 5′-ATCGCGCTTGGAATAGCTTA-3′, (SEQ ID NO: 10)5′-GACAGCCGTTCCAATGATCT-3′, (SEQ ID NO: 11) 5′-AGGCCGGGTAGAGATTGACT-3′,(SEQ ID NO: 12) 5′-CCTGCAGCACCAATCTGTTA-3′, (SEQ ID NO: 13)5′-CAGTGTTTATGGTGGCATCG-3′, (SEQ ID NO: 14) 5′-GGCATCGTGATAAGCCATTT-3′,(SEQ ID NO: 15) 5′-TGGCAGAGCTTGACATTGAC-3′, (SEQ ID NO: 16)5′-GCCGTTCTCTCAATCCACAT-3′, (SEQ ID NO: 17) 5′-ATACTGGGGCAGTGTCAAGG-3′,(SEQ ID NO: 18) 5′-TAACGTTCTTGCCAGGTTCC-3′, (SEQ ID NO: 19)5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20) 5′-ACAGGTTGTAGTTCGGCACC-3′,(SEQ ID NO: 21) 5′-CCAGGCACTTCAGATCCATT-3′, (SEQ ID NO: 22)5′-CTAGGCACAAACCAAACCGT-3′, (SEQ ID NO: 23) 5′-GATTGACGCCAGGGTGTACT-3′,(SEQ ID NO: 24) 5′-ATGTCTTCCCCATGAAGTGC-3′, (SEQ ID NO: 25)5′-CGCAGACAGACAACCAGCTA-3′, (SEQ ID NO: 26) 5′-TTGACCTCAATTCTTTGCCC-3′,(SEQ ID NO: 27) 5′-GACGTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-TCCGGCTTCTCGTACTGTCT-3′, (SEQ ID NO: 29) 5′-CTCTGTTTGGAACGCAACAA-3′,(SEQ ID NO: 30) 5′-GCCCCACCTCTTTTTAGTCC-3′, (SEQ ID NO: 31)5′-AGTCGAGCTTCAGGCAATGT-3′, (SEQ ID NO: 32) 5′-TGGTGTCTGAGTTGAGCAGG-3′,(SEQ ID NO: 33) 5′-TGAGTACAGTTCGACGTGGC-3′, (SEQ ID NO: 34)5′-TTGAGAGGAGCCTGACCACT-3′, (SEQ ID NO: 35) 5′-AGCTAAGGTGCTTGAGCTGC-3′,(SEQ ID NO: 36) 5′-ATGACGGTTCTTCCATCAGC-3′, (SEQ ID NO: 37)5′-ACATCCAAGAGTGGAAACCG-3′, (SEQ ID NO: 38) 5′-CGAGCTCTGCCTACCAATTC-3′,(SEQ ID NO: 39) 5′-GCAGGAGGAGAGTGGATGAC-3′, (SEQ ID NO: 40)5′-TTCTCCACTGGGGTTTTGTC-3′, (SEQ ID NO: 41) 5′-GGGTTAACAAAGGCAAACCA-3′,(SEQ ID NO: 42) 5′-CCCTGACCTACAGCTTCAG-3′, (SEQ ID NO: 43)5′-TAGTTCGCCTGTGTGAGCTG-3′, (SEQ ID NO: 44) 5′-TTTTAGCATATTGACAGCCCG-3′,(SEQ ID NO: 45) 5′-TTGATTGGACTGAAGAGGGC-3′, (SEQ ID NO: 46)5′-GCAATTGCTGTGAACCTGAA-3′, (SEQ ID NO: 47) 5′-GCTGAAGCTGTAGGTCAGGG-3′,(SEQ ID NO: 48) 5′-CTGGTTGTGCAGAGCAGAAG-3′, (SEQ ID NO: 49)5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50) 5′-GTCTCCTCTAACCTCTAGTCC-3′,(SEQ ID NO: 51) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′, (SEQ ID NO: 53)

More preferably, amplification is done with a pair of oligonucleotidesof the invention as primers, the pair of primers selected from the groupconsisting of

(a) 5′-CTGGATCGATGGAGAGGTGT-3′ (SEQ ID NO: 2) and5′-TCCGGTCTTTCCTCCTCTTT-3′ (SEQ ID NO: 3) (b) 5′-CTACCGTCAGCGATCTCTCC-3′(SEQ ID NO: 4) and 5′-TTCCTTTGCCAAATAGTCCG-3′ (SEQ ID NO: 5) (c)5′-GACGTCGGGTCATTTGAAGT-3′ (SEQ ID NO: 6) and 5′-ACTGCAATTCCAACACCACA-3′(SEQ ID NO: 7) (d) 5′-ATGTCCTGGATAACGCAAGG-3′ (SEQ ID NO: 8) and5′-CTCCTCCAACTGCGAGAAAC-3′ (SEQ ID NO: 9) (e) 5′-ATCGCGCTTGGAATAGCTTA-3′(SEQ ID NO: 10) and 5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO: 11) (f)5′-AGGCCGGGTAGAGATTGACT-3′ (SEQ ID NO: 12) and5′-CCTGCAGCACCAATCTGTTA-3′ (SEQ ID NO: 13) (g)5′-CAGTGTTTATGGTGGCATCG-3′ (SEQ ID NO: 14) and5′-GGCATCGTGATAAGCCATTT-3′ (SEQ ID NO: 15) (h)5′-TGGCAGAGCTTGACATTGAC-3′ (SEQ ID NO: 16) and5′-GCCGTTCTCTCAATCCACAT-3′ (SEQ ID NO: 17) (i)5′-ATACTGGGGCAGTGTCAAGG-3′ (SEQ ID NO: 18) and5′-TAACGTTCTTGCCAGGTTCC-3′ (SEQ ID NO: 19) (j)5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) and 5′ACAGGTTGTAGTTCGGCACC-3′(SEQ ID NO: 21) (k) 5′-CCAGGCACTTCAGATCCATT-3′ (SEQ ID NO: 22) and5′-CTAGGCACAAACCAAACCGT-3′ (SEQ ID NO: 23) (l)5′-GATTGACGCCAGGGTGTACT-3′ (SEQ ID NO: 24) and5′-ATGTCTTCCCCATGAAGTGC-3′ (SEQ ID NO: 25) (m)5′-CGCAGACAGACAACCAGCTA-3′ (SEQ ID NO: 26) and5′-TTGACCTCAATTCTTTGCCC-3′ (SEQ ID NO: 27) (n)5′-GACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) (o)5′-CTCTGTTTGGAACGCAACAA-3′ (SEQ ID NO: 30) and5′-GCCCCACCTCTTTTTAGTCC-3′ (SEQ ID NO: 31) (p)5′-AGTCGAGCTTCAGGCAATGT-3′ (SEQ ID NO: 32) and5′-TGGTGTCTGAGTTGAGCAGG-3′ (SEQ ID NO: 33) (q)5′-TGAGTACAGTTCGACGTGGC-3′ (SEQ ID NO: 34) and5′-TTGAGAGGAGCCTGACCACT-3′ (SEQ ID NO: 35) (r)5′-AGCTAAGGTGCTTGAGCTGC-3′ (SEQ ID NO: 36) and5′-ATGACGGTTCTTCCATCAGC-3′ (SEQ ID NO: 37) (s)5′-ACATCCAAGAGTGGAAACCG-3′ (SEQ ID NO: 38) and5′-CGAGCTCTGCCTACCAATTC-3′ (SEQ ID NO: 39) (t)5′-GCAGGAGGAGAGTGGATGAC-3′ (SEQ ID NO: 40) and5′-TTCTCCACTGGGGTTTTGTC-3′ (SEQ ID NO: 41) (u)5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) and 5′-CCCTGACCTACAGCTTCAG-3′(SEQ ID NO: 43) (v) 5′-TAGTTCGCCTGTGTGAGCTG-3′ (SEQ ID NO: 44) and5′-TTTTAGCATATTGACAGCCCG-3′ (SEQ ID NO: 45) (w)5′-TTGATTGGACTGAAGAGGGC-3′ (SEQ ID NO: 46) and5′-GCAATTGCTGTGAACCTGAA-3′ (SEQ ID NO; 47) (x)5′-GCTGAAGCTGTAGGTCAGGG-3′ (SEQ ID NO: 48) and5′-CTGGTTGTGCAGAGCAGAAG-3′ (SEQ ID NO: 49) (y)5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO: 51) (z)5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

Most preferably amplification is done using a pair of isolatedoligonucleotides selected from the group consisting of

(f) 5′-ATCGCGCTTGGAATAGCTTA-3′ (SEQ ID NO: 10) and5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO: 11) (j)5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) and5′-ACAGGTTGTAGTTCGGCACC-3′ (SEQ ID NO: 21) (n)5′-ACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) (u)5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) and 5′-CCCTGACCTACAGCTTCAG-3′(SEQ ID NO: 43) (y) 5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO: 51) (z)5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) and5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

Especially preferred pairs of isolated oligonucleotides for amplifyingWest Nile virus nucleic acid are

5′-ACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) and5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) 5′-GCCACCGGAAGTTGAGTAGA-3′(SEQ ID NO: 52) and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO: 53)

Amplified nucleic acid can be detected using a variety of detectiontechnologies well known in the art. For example, amplification productsmay be detected using agarose gel by performing electrophoresis withvisualization by ethidium bromide staining and exposure to ultraviolet(UV) light, by sequence analysis of the amplification product forconfirmation, or hybridization with an oligonucleotide probe.

Preferably, amplified nucleic acid is detected by hybridization with anoligonucleotide probe derived from the West Nile virus consensussequence or its complement. Probe sequences preferably are 10 to 50nucleotides long, more preferably 15 to 40 nucleotides long, mostpreferably 25-35 nucleotides long and are selected from the sequencethat is amplified by a pair of primers. Probes can be optionally labeledwith a detectable label.

Preferred probes include an isolated oligonucleotide selected from thegroup consisting of

(SEQ ID NO: 54) 5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′, (SEQ ID NO: 55)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, (SEQ ID NO: 56)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′, (SEQ ID NO: 58)5′-CAGGAGGACTGGGTTAACAAAGGCAAACCA-3′, and (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′.

More preferably the oligonucleotide probe is

(SEQ ID NO: 56) 5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′, or (SEQ ID NO: 59)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′.

Preferably, the oligonucleotide probe is labeled with a detectablelabel. The detectable label can be any molecule or moiety having aproperty or characteristic that is capable of detection, such as, forexample, radioisotopes, fluorophores, chemiluminophores, enzymes,colloidal particles, and fluorescent microparticles.

Probe sequences can be employed using a variety of methodologies todetect amplification products. Generally all such methods employ a stepwhere the probe hybridizes to a strand of an amplification product toform an amplification product/probe hybrid. The hybrid can then bedetected using labels on the primer, probe or both the primer and probe.Examples of homogeneous detection platforms for detecting amplificationproducts include the use of FRET (fluorescence resonance energytransfer) labels attached to probes that emit a signal in the presenceof the target sequence. “TaqMan” assays described in U.S. Pat. Nos.5,210,015; 5,804,375; 5,487,792 and 6,214,979 (each of which is hereinincorporated by reference) and Molecular Beacon assays described in U.S.Pat. No. 5,925,517 (herein incorporated by reference) are examples oftechniques that can be employed to detect nucleic acid sequences. Withthe “TaqMan” assay format, products of the amplification reaction can bedetected as they are formed or in a so-called “real time” manner. As aresult, amplification product/probe hybrids are formed and detectedwhile the reaction mixture is under amplification conditions.

Preferably, the PCR probes are TaqMan® probes that are labeled at the 5′end with a fluorophore and at the 3′-end with a quencher molecule.Suitable fluorophores and quenchers for use with TaqMan® probes aredisclosed in U.S. Pat. Nos. 5,210,015, 5,804,375, 5,487,792 and6,214,979 and WO 01/86001 (Biosearch Technologies). Preferred quenchersare Black Hole Quenchers disclosed in WO 01/86001.

In preferred embodiments of the invention, oligonucleotide primers areused to amplify West Nile virus nucleic acid in a test sample by PCRafter reverse transcription of the West Nile virus RNA to DNA. Amplifiednucleic acid is detected using a dual labeled “TaqMan” probe that islabeled with 5-carboxyfluorescein (FAM) at the 5′ end and a Black Holequencher as disclosed in WO 01/86001 at the 3′ end. Fluorescence isdetected using a fluorimeter.

The method of the invention is preferably performed using a set ofoligonucleotides selected from the group consisting of

(aa) 5′-ATCGCGCTTGGAATAGCTTA-3′, (SEQ ID NO: 10)5′-TGGATTTGGTCTCACCAGCACTCGGATGTT-3′ (SEQ ID NO: 54) optionally labeledwith a detectable label, and 5′-GACAGCCGTTCCAATGATCT-3′; (SEQ ID NO: 11)(bb) 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′ (SEQ ID NO: 55) optionally labeledwith a detectable label, and 5′-ACAGGTTGTAGTTCGGCACC-3′; (SEQ ID NO: 21)(cc) 5′-GACCTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′ (SEQ ID NO: 56) optionally labeledwith a detectable label, and 5′-TCCGGCTTCTCGTACTGTCT-3′; (SEQ ID NO: 29)(dd) 5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 42)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′ (SEQ ID NO: 57) optionally labeledwith a detectable label, and 5′-CCCTGACCTACAGCTTCAG-3′ (SEQ ID NO: 43)(ee) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50)5′-CAGGAGGACTGGGTTAACAAAGGCAAAGCA-3′ (SEQ ID NO: 58) optionally labeledwith a detectable label, and 5′-GTCTCCTCTAACCTCTAGTCC-3′; (SEQ ID NO:51) (ff) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′ (SEQ ID NO: 59) optionally labeledwith a detectable label, and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO:53)

More preferably, the method of the invention is performed using eitherof the following sets of oligonucleotides

(cc) 5′-GACCTCCCAGAATTAGAGCGC-3′, (SEQ ID NO: 28)5′-ACGGGTTCACTACTACTGCATCTAGAGACA-3′ (SEQ ID NO: 56) optionally labeledwith a detectable label, and 5′-TCCGGCTTCTCGTACTGTCT-3′; (SEQ ID NO: 29)(ff) 5′-GCCACCGGAAGTTGAGTAGA-3′, (SEQ ID NO: 52)5′-ATCACTTCGCGGCTTTGTTCACCCAGTCCT-3′ (SEQ ID NO: 59) optionally labeledwith a detectable label, and 5′-GAGACGGTTCTGAGGGCTTAC-3′. (SEQ ID NO:53)

In each of the foregoing sets of oligonucleotides, the first and thirdoligonucleotides are used as primers and the middle oligonucleotide isused as a probe for detecting amplified nucleic acid. The probe isoptionally labeled with a detectable label.

The oligonucleotides of the invention can also be used to detect WestNile virus using methods and techniques that are known in the art thatare not based on nucleic acid amplification.

Nucleic acid hybridization can be done using techniques and conditionsknown in the art. Specific hybridization conditions will depend on thetype of assay in which hybridization is used. Hybridization techniquesand conditions can be found, for example, in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel etal., eds. (1995) Current Protocols in Molecular Biology, Chapter 2(Greene Publishing and Wiley-Interscience, New York) and Sambrook et al.(1989) Molecular Cloning. A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Hybridization of nucleic acid may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified. Alternatively, stringency conditions can be adjustedto allow some mismatching in sequences so that lower degrees ofsimilarity are detected.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at37.degree. C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Theduration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours, or less depending on the assay format.

It should be noted that the oligonucleotides of the invention can beused as primers or probes, depending on the intended use or assayformat. For example, an oligonucleotide used as a primer in one assaycan be used as a probe in another assay. The grouping of theoligonucleotides into primer pairs and primer/probe sets reflectscertain preferred embodiments of the invention. However, the use ofother primer pairs comprised of forward and reverse primers selectedfrom different preferred primer pairs is specifically contemplated.

The term “test sample” as used herein, means anything designated fortesting for the presence of West Nile virus and/or West Nile virusnucleic acid. The test sample is, or can be derived from any biologicalsource, such as for example, blood, blood plasma, cell cultures, tissuesand mosquito samples. The test sample can be used directly as obtainedfrom the source, or following a pre-treatment to modify the character ofthe sample. Thus, the test sample can be pre-treated prior to use by,for example, preparing plasma from blood, disrupting cells or viralparticles, preparing liquids from solid materials, diluting viscousfluids, filtering liquids, distilling liquids, concentrating liquids,inactivating interfering components, adding reagents, and purifyingnucleic acid.

The oligonucleotides of the invention can be made with standardmolecular biology techniques known in the art and disclosed in manualssuch as Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989) orconventional nucleotide phosphoramidite chemistry and commerciallyavailable synthesizer instruments. The oligonucleotides of the inventioncan be DNA or RNA. The invention is also directed to the RNA equivalentsof the oligonucleotides of the invention and their complements.

Another aspect of the invention provides a method of identifying primersfor detection of a nucleic acid sequence, which method comprises thesteps of (a) providing a nucleic acid sequence at least 1000 bases inlength; (b) dividing the nucleic acid sequence into non-overlappingsegments approximately 500 bases in length starting from one end of thesequence; and (c) selecting forward and reverse primers each about 15-25bases in length from the sequence of at least one segment and/or itscomplement, wherein the forward and reverse primers are selected to havenon-overlapping sequences and produce an amplicon having from about 50to 200 bases.

The method of the invention can be used for identifying primers fordetection of any nucleic acid sequence at least about 1,000 bases inlength, including the entire genome of an organism such as a virus.Unlike prior methods where portions of a known gene or other location inthe sequence were used to identify primers, the method of the inventionsystematically covers the entire sequence without regard to open readingframes or other landmarks in the sequence. The method can be used withany type of sequence including native or consensus sequences. Thenucleic acid sequence is divided into non-overlapping segmentsapproximately 500 bases long. For sequences that are not multiples of500, the length of the segments can be adjusted to take into accountadditional bases, or the additional bases at the end can be used as ashorter segment. One or more sets of primers are selected from at leastone segment and/or its complement, preferably using primer designsoftware such as 3 Primer 3 PCR primer design software (WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). Preferably, one ormore sets of primers are selected from each of the segments. Primerdesign takes generally takes into account factors such as primer length,melting temperature (T_(m)), specificity, complementary primersequences, G/C content, polypyrimidine (T,C) or polypurine (A,G)stretches, and the assay format and conditions under which the primerswill be used. The primer sets can be tested for their ability to amplifythe segment from which they were selected using the same assay formatand conditions.

With regard to the West Nile virus, a 10944 bp region of the consensussequence in FIG. 1 that showed the highest sequence conservation fromthe eight full-length US WNV isolates was selected for PCR amplificationprimer design. This region of homology was systematically broken intoapproximately 500 bp sections, and within each section 1 pair ofamplification primers were selected using Primer 3 PCR primer designsoftware (Whitehead Institute for Biomedical Research, Cambridge,Mass.). Default settings for primer design using Primer 3 were optimizedfor 100-150 bp amplicons, primer T_(m) (melting temperature) in the55-60° C. range, and G/C content optimized for dual-labeled fluorescentprobe-based assays (“TaqMan” assays).

Another aspect of the invention provides methods for detecting West Nilevirus in a test sample comprising the steps of amplifying West NileVirus nucleic acid in said test sample; and detecting amplified nucleicacid, wherein detection of amplified nucleic acid indicates the presenceof West Nile virus in said test sample, wherein the method uses at leastone oligonucleotide identified according to the method discussed aboveto amplify or detect West Nile virus nucleic acid.

Utilization of the West Nile virus consensus sequence shown in FIG. 1for selection of primers and probes resulted in identification ofoligonucleotides with higher sequence homology to North American orIsraeli West Nile virus isolates than West Nile virus infectionsoriginating outside North America or Israel.

An additional aspect of the invention thus provides an isolatedoligonucleotide comprising from about 15 to about 75 contiguous bases ofthe consensus sequence or its complement, more preferably from about 15to about 60 contiguous bases, most preferably from about 15 to about 30continuous bases of the consensus sequence or its complement, whereinthe oligonucleotide bonds with greater affinity to nucleic acid fromNorth American or Israeli West Nile virus isolates than West Nile virusisolates originating outside North America or Israel.

Lanciotti et al., Virology, 298: 96-105, 2002 discloses a phylogeneticanalysis of West Nile Virus Strains isolated from the United States,Europe and the Middle East. The phylogenetic trees constructed byLanciotti et al. revealed the presence of two genetic lineages of WestNile viruses. Lineage 1 West Nile viruses have been isolated from thenortheastern United States, Europe, Israel, Africa, India, Russia andAustralia. Lineage 2 West Nile viruses have been isolated only insub-Saharan Africa and Madagascar. The lineage 1 viruses were furthersubdivided into three monophyletic clades. All of the isolatesoriginating from the United States and Israel were found to be closelyrelated to each other and were placed into the same clade, referred toas the US/Israel clade. Accession numbers for the American isolates usedfor phylogenetic analysis by Lanciotti et al. and by the Applicants forconstruction of the consensus sequence of FIG. 1 are in Example 1. Theaccession number of the isolate from an Israeli stork is GeneBankaccession no. AY033389. Lanciotti et al. found that isolates from theUnited States and Israel had greater than 99% nucleotide sequenceidentity and amino acid identity. Isolates originating from other partsof the world had less nucleotide sequence identity (96.5% or less withthe U.S. and Israeli isolates, but in many cases the amino acid sequenceidentity was still greater than 99%.

The invention further provides test kits comprising at least oneoligonucleotide of the invention. Often, test kits contain one or morepairs of oligonucleotides such as the primer pairs disclosed herein, orone or more oligonucleotide sets as disclosed herein. The assay kit canfurther comprise the four-deoxynucleotide phosphates (dATP, dGTP, dCTP,dTTP) and an effective amount of a nucleic acid polymerizing enzyme. Anumber of enzymes are known in the art which are useful as polymerizingagents. These include, but are not limited to E. coli DNA polymerase I,Klenow fragment, bacteriophage T7 RNA polymerase, reverse transcriptase,and polymerases derived from thermophilic bacteria, such as Thermusaquaticus. The latter polymerases are known for their high temperaturestability, and include, for example, the Taq DNA polymerase I. Otherenzymes such as Ribonuclease H can be included in the assay kit forregenerating the template DNA. Other optional additional components ofthe kit include, for example, means used to label the probe and/orprimer (such as a fluorophore, quencher, chromogen, etc.), and theappropriate buffers for reverse transcription, PCR, or hybridizationreactions. Usually, the kit also contains instructions for carrying outthe methods.

EXAMPLES Example 1 West Nile Virus (WNV) Sequence Analysis: ConsensusSequence

WNV has a single strand positive-polarity RNA genome of approximately 11kb. GenBank searches resulted in the identification of eightapproximately full-length WNV genome sequences from isolates identifiedin the United States (GenBank accession numbers: AF196835, AF260967,AF202541, AF206518, AF404753, AF404754, AF404755 and AF404756). Sequencealignment and comparisons were performed using Vector Nti Suite 7.0(InforMax, Frederick, Md., USA) and a consensus sequence was derivedfrom the analysis of these eight sequences (entire WNV consensussequence is listed in FIG. 1). The currently known US isolates of WNVhave high homology at the nucleotide level, most likely the result of arecent “founder effect” infection of the virus in the US. WNV sequencecomparison to European and other more ancestral isolates to the USstrains reveals a much higher diversity at the nucleotide level.

Example 2 2.1 PCR Amplification Primer Design

From the WNV consensus sequence, a 10944 bp region of highest sequenceconservation from the eight full-length US WNV isolates was selected forPCR amplification primer design. This region of homology wassystematically broken into 500 bp sections, and within each section 1pair of amplification primers were selected using Primer 3 PCR primerdesign software (Whitehead Institute for Biomedical Research, Cambridge,Mass.). Default settings for primer design using Primer 3 were optimizedfor 100-150 bp amplicons, primer T_(m) (melting temperature) in the55-60° C. range, and G/C content optimized for dual-labeled fluorescentprobe-based assays (Applied Biosystems, Foster City, Calif.).

Primer sequences identified for WNV amplification were chemicallysynthesized. Each of the individual amplification primer pairs wereassayed for the ability to amplify WNV RNA using reverse transcriptionand polymerase chain reaction (RT-PCR; Brilliant™ Plus Single StepQRT-PCR system, Stratagene, La Jolla, Calif., USA). Reactions were setupusing each of the thirty-one WNV amplification primer pairs along with ano template negative PCR control. After RT-PCR reactions were completed,5 μL of the total 50 μL reaction volume was used for electrophoresis ona 4-20% polyacrylamide gel to determine if the RT-PCR had beensuccessful. PCR amplicons of the predicted size were observed in 28 of31 reactions performed.

Primers, excluding the primer pairs that produced no amplicon, are shownin Table 1.

TABLE 1 (a) WNVamplF 5′-CTGGATCGATGGAGAGGTGT-3′ (SEQ ID NO: 2) WNVamplR5′-TCCGGTCTTTCCTCCTCTTT-3′ (SEQ ID NO: 3) (b) WNVamp3F5′-CTACCGTCAGCGATCTCTCC-3′ (SEQ ID NO: 4) WNVamp3R5′-TTCCTTTGCCAAATAGTCCG-3′ (SEQ ID NO: 5) (c) WNVamp4F5′-GACGTCGGGTCATTTGAAGT-3′ (SEQ ID NO: 6) WNVamp4R5′-ACTGCAATTCCAACACCACA-3′ (SEQ ID NO: 7) (d) WNVamp5F5′-ATGTCCTGGATAACGCAAGG-3′ (SEQ ID NO: 8) WNVamp5R5′-CTCCTCCAACTGCGAGAAAC-3′ (SEQ ID NO: 9) (e) WNVamp6F5′-ATCGCGCTTGGAATAGCTTA-3′ (SEQ ID NO: 10) WNVamp6R5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO: 11) (f) WNVamp7F5′-AGGCCGGGTAGAGATTGACT-3′ (SEQ ID NO: 12) WNVamp7R5′-CCTGCAGCACCAATCTGTTA-3′ (SEQ ID NO: 13) (g) WNVamp8F5′-CAGTGTTTATGGTGGCATCG-3′ (SEQ ID NO: 14) WNVamp8R5′-GGCATCGTGATAAGCCATTT-3′ (SEQ ID NO: 15) (h) WNVamp9F5′-TGGCAGAGCTTGACATTGAC-3′ (SEQ ID NO: 16) WNVamp9R5′-GCCGTTCTCTCAATCCACAT-3′ (SEQ ID NO: 17) (i) WNVamp10F5′-ATACTGGGGCAGTGTCAAGG-3′ (SEQ ID NO: 18) WNVamp10R5′-TAACGTTCTTGCCAGGTTCC-3′ (SEQ ID NO: 19) (j) WNVamp11F5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) WNVamp12R5′-ACAGGTTGTAGTTCGGCACGT-3′ (SEQ ID NO: 21) (k) WNVamp12F5′-CCAGGCACTTCAGATCCATT-3′ (SEQ ID NO: 22) WNYamp12R5′-CTAGGCACAAACCAAACCGT-3′ (SEQ ID NO: 23) (l) WNYampl3F5′-GATTGACGCCAGGGTGTACT-3′ (SEQ ID NO: 24) WNVamp13R5′-ATGTCTTCCCCATGAAGTGC-3′ (SEQ ID NO: 25) (m) WNVamp14F5′-CGCAGACAGACAACCAGCTA-3′ (SEQ ID NO: 26) WNVamp14R5′-TTGACCTCAATTCTTTGCCC-3′ (SEQ ID NO: 27) (n) WNVamp15F (2)5′-GACGTCCCAGAATTAGAGCGC-3′ (SEQ ID NO: 28) WNVamp15R (2)5′-TCCGGCTTCTCGTACTGTCT-3′ (SEQ ID NO: 29) (o) WNVamp16F5′-CTCTGTTTGGAACGCAACAA-3′ (SEQ ID NO: 30) WNVamp16R5′-GCCCCACCTCTTTTTAGTCC-3′ (SEQ ID NO: 31) (p) WNVamp17F5′-AGTCGAGCTTCAGGCAATGT-3′ (SEQ ID NO: 32) WNVamp17R5′-TGGTGTCTGAGTTGAGCAGG-3′ (SEQ ID NO: 33) (q) WNVamp18F5′-TGAGTACAGTTCGACGTGGC-3′ (SEQ ID NO: 34) WNVamp18R5′-TTGAGAGGAGCCTGACCACT-3′ (SEQ ID NO: 35) (r) WNVamp19F5′-AGCTAAGGTGCTTGAGCTGC-3′ (SEQ ID NO: 36) WNVamp19R5′-ATGACGGTTCTTCCATCAGC-3′ (SEQ ID NO: 37) (s) WNVamp20F5′-ACATCCAAGAGTGGAAACCG-3′ (SEQ ID NO: 38) WNVamp20R5′-CGAGCTCTGCCTACCAATTC-3′ (SEQ ID NO: 39) (t) WNVamp21F5′-GCAGGAGGAGAGTGGATGAC-3′ (SEQ ID NO: 40) WNVamp21R5′-TTCTCCACTGGGGTTTTGTC-3′ (SEQ ID NO: 41) (u) WNVamp22F5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) WNVamp22R5′-CCCTGACCTACAGCTTCAG-3′ (SEQ ID NO: 43) (v) WNVamp23A-F5′-TAGTTCGCCTGTGTGAGCTG-3′ (SEQ ID NO: 44) WNVamp23A-R5′-TTTTAGCATATTGACAGCCCG-3′ (SEQ ID NO: 45) (w) WNVamp23C-F5′-TTGATTGGACTGAAGAGGGC-3′ (SEQ ID NO: 46) WNVamp23C-R5′-GCAATTGCTGTGAACCTGAA-3′ (SEQ ID NO: 47) (x) WNVamp24A-F5′-GCTGAAGCTGTAGGTCAGGG-3′ (SEQ ID NO: 48) WNVamp24A-R5′-CTGGTTGTGCAGAGCAGAAG-3′ (SEQ ID NO: 49) (y) WNVamp24B-F5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) WNVamp24B-R5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO: 51) (z) WNVamp24C-F5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) WNVamp24C-R5′-GAGACGGTTCTGAGGGCTTAC-3′ (SEQ ID NO: 53)

2.2 Probe Design for Fluorescence-based PCR Detection of WNV

Of the twenty-eight positive PCR amplification primer pairs identifiedwithin the consensus sequence of the WNV genome (FIG. 1), six sets werechosen for consideration in development of fluorescent detection ofnucleic acid based on the ability to amplify WNV target at lowconcentrations in solution (Table 2). For this purpose, the sequences ofthese six WNV amplicons were evaluated using PrimerQuest^(SM) software(Integrated DNA Technologies, Coralville, Iowa, USA) to identify optimalfluorescent probes that would hybridize within the WNV amplicon. Defaultsettings for running the software were optimum probe size=30 nt; optimumprobe Tm=70° C.; and optimum probe GC %=50. Probe positioning wasprioritized to be within a maximum of 20 nucleotides 3′ of theamplification primer, and both strands were analyzed to identify thesingle optimal probe that could be identified within each of the six WNVamplicons. Probes selected are shown in Table 2.

Fluorescent WNV probes containing 5-carboxyfluorescein (FAM) at the 5′end and a Black Hole Quencher (Bhquencher; BioSearch Technologies,Novato, Calif., USA) at the 3′ end were synthesized. Each of the sixamplification primer pair/fluorescent probe combinations (which areshown in Table 2) were tested by RT-PCR (Brilliant™ Plus Single StepQRT-PCR system, Stratagene, La Jolla, Calif., USA), for detection of WNVin diluted concentrations of target in normal human plasma (NHP). Targetwas diluted 1:20, 1:100, 1:200, 1:1,000, 1:2,000 and 1:10,000. All sixamplification primer pair/fluorescent probe combinations were capable ofdetecting WNV in human plasma.

TABLE 2 (e) WNVamp6F 5′-ATCGCGCTTGGAATAGCTTA-3′ (SEQ ID NO: 10) WNVamp6Probe 5′FAM-TGGATTTGGTCTCACCAGCA- (SEQ ID NO: 54)CTCGGATGTT-3′BHquencher WNVamp6R 5′-GACAGCCGTTCCAATGATCT-3′ (SEQ ID NO:11) (j) WNVamp11F 5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) WNVamp11Probe 5′FAM-ACATCCGCAGTGCCCAGAGAA- (SEQ ID NO: 55)CATAATGGA-3′BHquencher WNVamp11R 5′ACAGGTTGTAGTTCGGCACC-3′ (SEQ ID NO:21) (n) WNVamp15F (2) 5′-TCCCAGAATTAGAGCGCGCGC-3′ (SEQ ID NO: 28)WNVamp15 ProbeR 5′FAM-ACGGGTTCACTACTACTGC- (SEQ ID NO: 56)ATCTAGAGACA-3′BHquencher WNVamp15R (2) 5′-TCGGTCCGGCTTCTCGTACT-3′ (SEQID NO: 29) (u) WNVamp22F 5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42)WNVamp22 Probe 5′FAM-CCGGTAATGGTGTTAAACCAG- (SEQ ID NO: 57)GGCGAAAGGA-3′BHquencher WNVamp22R 5′-CCCTGACCTACAGCTTCAG-3′ (SEQ ID NO:43) (y) WNVamp24B-F 5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) WNVamp24BProbe 5′FAM-CAGGAGGACTGGGTTAACAAAGGC- (SEQ ID NO: 58)AAACCA-3′BHquencher WNVamp24B-R 5′-GTCTCCTCTAACCTCTAGTCC-3′ (SEQ ID NO:51) (z) WNVamp24C-F 5′-GCCACCGGAAGTTGAGTAGA-3′ (SEQ ID NO: 52) WNVamp24CProbe 5′FAM-ATCACTTCGCGGCTTTGTTCA- (SEQ ID NO: 59)CCCAGTCCT-3′BHquencher WNVamp24C-R 5′-GAGACGGTTCTGAGGGCTTAC-3′ (SEQ IDNO: 53)

Example 3 3.1 Optimization of WNV Nucleic Acid Isolation from HumanPlasma

Nucleic acid isolations were performed on normal human plasma that hadbeen spiked with known concentrations of WNV RNA using a Guanidineisothyocyanate lysis/alcohol precipitation procedure (AmpliScreen™Multiprep Specimen Processing Procedure, Roche Diagnostics, Basel,Switzerland). A polyacrylamide carrier was added prior to isopropanolprecipitation of RNA. One-mL volumes of plasma were utilized perisolation, and precipitated nucleic acid was suspended in elution bufferin a total volume of 200 μL. 25% of the total isolation volume (50 μL)was utilized for each RT-PCR reaction.

3.2 Optimization of RT-PCR Conditions for the Detection of WNV fromHuman Plasma

The Brilliant™ Plus Single Step QRT-PCR system (Stratagene, La Jolla,Calif., USA) with minor modifications from the vendor's recommendationswas utilized for nucleic acid amplification. Bovine serum albumin (BSA)was added to RT-PCR reactions. Real-time PCR was performed on anABI7900HT DNA Detection System (Applied Biosystems, Foster City, Calif.,USA). RT-PCR thermal-cycling conditions utilized were: 45° C. for 30minutes; 95° C. for 8 minutes; then 60 cycles alternating between 95° C.for 15 seconds and 55° C. for 1 minute.

1. An isolated oligonucleotide consisting of (a) R₁—N—R₂ wherein N is anoligonucleotide selected from the group consisting of (SEQ ID NO: 20)5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 21) 5′-ACAGGTTGTAGTTCGGCACC-3′,(SEQ ID NO: 42) 5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 43)5′-CCCTGACCTACAGCTTCAG-3′, (SEQ ID NO: 50) 5′-GGAGAGTGCAGTCTGCGATA-3′,(SEQ ID NO: 51) 5′-GTCTCCTCTAACCTCTAGTCC-3′, (SEQ ID NO: 55)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′,

R₁ is an oligonucleotide sequence of 0-20 contiguous bases of the WestNile virus consensus sequence shown in FIG. 1 (SEQ ID NO:1) immediatelyupstream of the 5′ end of N in said consensus sequence covalently linkedto N at the 5′ end, provided that when N is complementary to saidconsensus sequence, R₁ is selected from the complement of said consensussequence; and R₂ is an oligonucleotide sequence of 0-20 contiguous basesof said consensus sequence immediately downstream of the 3′-end of N insaid consensus sequence covalently linked to N at the 3′ end, providedthat when N is complementary to said consensus sequence, R₂ is selectedfrom the complement of said consensus sequence; (b) an isolated fragmentof N as defined in (a) wherein said fragment is 10-19bases in length;(c) R₁—X—R₂, wherein X is at least 10 contiguous bases of N as definedin (a), and R₁ and R₂ are as defined in (a), wherein when R₁ is present,R₂ is absent and X is selected such that the base at the 5′-end of X isthe same as the base at the 5′-end of N; and when R₂ is present, R₁ isabsent and X is selected such that the base at the 3′-end of X is thesame as the base at the 3′-end of N; (d) an isolated oligonucleotidewhich has at least 90% sequence identity with an oligonucleotide of (a),(b) or (c) and binds to SEQ ID NO: 1 or its complementary sequence; or(e) an isolated oligonucleotide which is the full-length complement of(a), (b), (c) or (d).
 2. The oligonucleotide of claim 1 wherein saidoligonucleotide consists of an oligonucleotide selected from the groupconsisting of 5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20)5′-ACAGGTTGTAGTTCGGCACC-3′, (SEQ ID NO: 21) 5′-GGGTTAACAAAGGCAAACCA-3′,(SEQ ID NO: 42) 5′-CCCTGACCTACAGCTTCAG-3′, (SEQ ID NO: 43)5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′. (SEQ ID NO: 51)


3. The oligonucleotide of claim 1 wherein said oligonucleotide consistsof an oligonucleotide selected from the group consisting of (SEQ ID NO:55) 5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′, and (SEQ ID NO: 57)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′.


4. The oligonucleotide of claim 3 wherein said oligonucleotide furthercomprises a detectable label.
 5. The oligonucleotide of claim 4 whereinsaid detectable label is a fluorescent molecule attached at the 5′ end.6. The oligonucleotide of claim 5 further comprising a quencher moleculeattached at the 3′ end.
 7. A pair of isolated oligonucleotide sequenceswherein said pair is selected from the group consisting of (j)5′-GGCTGAAGCACTGAGAGGAC-3′ (SEQ ID NO: 20) and5′-ACAGGTTGTAGTTCGGCACC-3′ (SEQ ID NO: 21) (u)5′-GGGTTAACAAAGGCAAACCA-3′ (SEQ ID NO: 42) and 5′-CCCTGACCTACAGCTTCAG-3′(SEQ ID NO: 43) and (y) 5′-GGAGAGTGCAGTCTGCGATA-3′ (SEQ ID NO: 50) and5′-GTCTCCTCTAACCTCTAGTCC-3′. (SEQ ID NO: 51)


8. A set of oligonucleotides selected from the group consisting of (bb)5′-GGCTGAAGCACTGAGAGGAC-3′, (SEQ ID NO: 20)5′-ACATCCGCAGTGCCCAGAGAACATAATGGA-3′ (SEQ ID NO: 55) optionally labeledwith a detectable label, and 5′-ACAGGTTGTAGTTCGGCACC-3′; (SEQ ID NO: 21)(dd) 5′-GGGTTAACAAAGGCAAACCA-3′, (SEQ ID NO: 42)5′-CCGGTAATGGTGTTAAACCAGGGCGAAAGGA-3′ (SEQ ID NO: 57) optionally labeledwith a detectable label, and 5′-CCCTGACCTACAGCTTCAG-3′ (SEQ ID NO: 43)and (ee) 5′-GGAGAGTGCAGTCTGCGATA-3′, (SEQ ID NO: 50)5′-CAGGAGGACTGGGTTAACAAAGGCAAAGCA-3′ (SEQ ID NO: 58) optionally labeledwith a detectable label, and 5′-GTCTCCTCTAACCTCTAGTCC-3′. (SEQ ID NO:51)


9. A method of detecting West Nile virus in a test sample comprising thesteps of amplifying West Nile Virus nucleic acid in said test sample;and detecting amplified nucleic acid, wherein detection of amplifiednucleic acid indicates the presence of West Nile virus in said testsample, wherein said method uses at least one oligonucleotide of claim 1to amplify or detect West Nile virus nucleic acid.
 10. A method ofdetecting West Nile Virus in a test sample comprising the steps of:amplifying West Nile Virus nucleic acid in a test sample using at leastone oligonucleotide of claim 2; and detecting amplified nucleic acid,wherein detection of amplified nucleic acid indicates the presence ofWest Nile virus in said test sample.
 11. A method of detecting West NileVirus in a test sample comprising the steps of: amplifying West NileVirus nucleic acid in said test sample; and detecting amplified nucleicacid using an oligonucleotide of claim 3, wherein detection of amplifiednucleic acid indicates the presence of West Nile virus in said testsample.
 12. A method of detecting West Nile Virus in a test samplecomprising the steps of: amplifying West Nile Virus nucleic acid in saidtest sample containing such virus using a pair of oligonucleotides ofclaim 7; and detecting amplified nucleic acid, where in detection ofamplified nucleic acid indicates the presence of West Nile virus in saidtest sample.
 13. A method of detecting West Nile Virus in a test samplecomprising the steps of: amplifying West Nile Virus nucleic acid in saidtest sample; and detecting amplified nucleic acid, wherein detection ofamplified nucleic acid indicates the presence of West Nile virus in saidtest sample, wherein said amplifying step and said detecting step areperformed with a set of claim
 8. 14. A method of detecting West NileVirus in a test sample comprising the steps of hybridizing at least oneoligonucleotide of claim 1 with West Nile virus nucleic acid in a testsample; and detecting hybridization of said at least one oligonucleotideof claim 1 with West Nile virus nucleic acid.
 15. The method of any oneof claims 9-13 and 14 wherein said test sample comprises human bloodplasma.
 16. A test kit comprising at least one oligonucleotide of claim1.